Nonalcoholic steatohepatitis (NASH) is the most common liver disease among veterans and is characterized by hepatic steatosis, sterile inflammation and liver damage [1]. NASH is associated with the metabolic syndrome, and can progress to cirrhosis and cancer. The mechanisms governing NASH development are not known, and currently there is no effective therapy. We have shown that the DNA receptor TLR9 (TOLL like receptor 9) is required for acute sterile inflammation in the liver and pancreas [2-4]. Here we propose to examine role of cell-free DNA (cfDNA) in TLR9 activation and its importance in murine and human NASH. Our hypothesis is that increased levels of mitochondrial DNA from hepatocytes contributes to the development of NASH via TLR9 activation on Kupffer cells, monocytes and hepatocytes. We will focus on identifying the cellular source and biochemical (oxidation) status of cfDNA, and the functional role of cfDNA in NASH. Our data will support the progression to clinical trials of a TLR9 antagonist that has already been safely used in humans. Preliminary data: i) Plasma levels of cfDNA are higher in mice fed a HFD and in obese patients with NASH, and the cfDNA is of mitochondrial origin. ii) Plasma from NASH subjects has an increase in intact mitochondria (mt), and the majority are in microparticles (MP). iii) Plasma DNA from mice with NASH is a potent ligand for TLR9. iv) Increased ability of plasma from NASH patients to activate a TLR9 reporter cell line is significantly reduced when MP are removed. v) Increase in oxidation state of mtDNA in plasma from patients with NASH. vi) Total body deletion of TLR9 results in less NASH in a HFD model of NASH. vii) Selective deletion of TLR9 on macrophages results in less NASH. viii) Hepatocytes mtDNA from mice fed a HFD has a greater degree of oxidation and a greater ability to stimulate inflammatory, but not anti-inflammatory pathway. ix) The TLR9 antagonist IRS954 protects mice against NASH. Aim 1: Identify the cellular origin and biochemical characteristics of DNA responsible for TLR9 activation in NASH. Aim 2: Identify the cell specific effects of TLR9 activation on a) monocytes and Kupffer cells and b) hepatocytes. Aim 3: Identify the role of mtDNA oxidation on the selective activation of pro-inflammatory pathways, and test a clinically available TLR9 antagonist in a mouse model of NASH. This grant will identify the cellular origins of the elevated the TLR9 ligand plasma cfDNA in NASH, and identify if the development of NASH increases the ability of DNA to function as a ligand for TLR9. It will further establish the role of TLR9 activation on KC, monocytes, and hepatocytes in the development of HFD NASH. Finally demonstration of the efficacy of a clinically available TLR9 antagonist in a mouse model of NASH will allow for the rational progression to clinical trials.